Despite the progress of the last few years, substantial gaps in our understanding of nucleoside resistance remain. The problem of dual resistance to zidovudine (AZT) and lamivudine (3TC) is of particular interest because viruses that are dually resistant to these two drugs show diminished susceptibility to other members of the nucleoside class. Great strides have been made in understanding the biochemical basis of resistance to the thymidine analogs, but the biochemical basis of dual resistance remains poorly understood. Similarly, the effects of novel 3TC resistance mutations such as the 44D/1181 cluster and associated polymorphisms on reverse transcriptase (RT) kinetics and viral fitness have not been studied in detail. In addition, the effects of AZT and 3TC resistance mutations on the kinetics of RT inhibition by carbovir triphosphate and PMPA diphosphate have not been studied. Specific aims of this proposal include: 1) To study steady-state and pre-steady-state kinetics of RT from AZT/3TC-resistant biological clones of HIV-1; 2) To determine the role of pyrophosphorolysis in AZT/3TC dual resistance through primer unblocking studies performed in Collaboration with Dr. Mathias Gotte of McGill University; 3) To determine the effect of RT polymorphisms associated with dual AZT/3TC resistance on the relative fitness of dually resistant isolates; 4) To extend these studies to carbovir and PMPA; and 5) to explore the effect of other "AZT-sensitizing" mutations (such as those encoding resistance to didanosine [L74V] and nevirapine [Y181C] on pyrophosphorolysis and primer unblocking. Better understanding of the virologic, genetic, and biochemical aspects of AZT/3TC dual resistance will help direct therapy and may lead to better therapeutic strategies in the future.